WO1999007350A2

WO1999007350A2 - Chemo-protection

Info

Publication number: WO1999007350A2
Application number: PCT/EP1998/004679
Authority: WO
Inventors: Manfred Kubbies
Original assignee: Roche Diagnostics Gmbh
Priority date: 1997-08-07
Filing date: 1998-07-25
Publication date: 1999-02-18
Also published as: WO1999007350A3; AU9156498A

Abstract

The invention relates to the use of L-cysteine and/or derivatives thereof for producing a pharmaceutical composition to selectively protect non-malignant cells, and to an extracorporeal method for selectively protecting non-malignant cells during a treatment to induce the cell death of malignant cells.

Description

Chemoprotection

The invention relates to the use of L-cysteine or / and derivatives thereof for the production of a pharmaceutical composition for the selective protection of non-malignant cells and an extracorporeal method for the selective protection of non-malignant cells during a therapy for inducing cell death of malignant cells.

Malignant tumors are serious diseases that are often not confined to one location. The formation of metastases can lead to an infection of various tissues and organs of a body. Malignant diseases not only significantly reduce a patient's quality of life, they very often have a fatal outcome. For this reason, great efforts have been made for many years to develop improved drugs and treatment methods that reduce the mortality rate or at least improve the quality of life and increase life expectancy.

Chemotherapy is a frequently used form of therapy for the treatment of malignant diseases. The cytostatics known today include classes of substances such as alkylating agents, antimetabolites, natural products, antibiotics and hormones. Commonly used substances are alkylating agents, which are also referred to as clastogens and lead to DNA cross-linking, such as cis-platinum (CDDP) or carbo-platinum.

Although these therapeutic agents have a high cytotoxicity for tumor cells, they can often only be administered in a therapeutically suboptimal dosage due to the considerable side effects associated with their use. The side effects are due to the often insufficiently specific mode of action of these agents. When used, malignant cells are not only attacked and rendered harmless, but healthy cells are also damaged. In addition, chronic therapy, such as renal insufficiency, can occur after such therapy. Even chemotherapy-induced malignancies, such as leukemia, are observed. As a result, complete confirmation of tumors is not successful in many cases. In particular, the haematopoietic toxicity of many chemotherapeutic agents in chemotherapy is the dose-limiting factor (Civin, C. L, Csore, SD, J. Hematotherapy 2 (1993) 137-144).

The cells that are important for blood formation are the hematopoietic stem cells in the bone marrow. Only about every hundred thousandth cell in the bone marrow is an undifferentiated hematopoietic stem cell that is able to duplicate and generate daughter cells that differentiate into hematopoietic lines. Damage to these cells by chemotherapeutic agents is therefore particularly serious for the organism.

Cis-platinum is a cytostatic used for various malignant tumors and, like practically all chemotherapy drugs, has significant side effects. Kubbies, M., et al, Br. J. Cancer 64 (1991) 847-849, were able to show that the cytotoxic effect of the cis-platinum is due to the intervention in the Gi / S phase of the cell cycle. Studies on peripheral blood lymphocytes (PBL) have shown that this effect can be partially offset by the use of glutathione.

In addition to the classic systemic treatment of a patient with chemotherapy drugs, extracorporeal treatment methods are also used today. Tissue or cells containing malignant cells are removed from the body. The removed preparation is then incubated with cytotoxic substances in a further process step in order to eliminate the malignant cells. The healthy cells are separated and, if necessary, cultivated to obtain a larger number of healthy cells, which are then re-implanted in the body. This method is used for example in bone marrow tumors and is referred to as "purging". Even with such methods, however, there are the above-mentioned side effects when administering cytotoxic substances and consequently damage to healthy cells.

There was therefore a need to find agents which reduce the side effects of known substances which induce cell death in malignant cells without reducing the effectiveness of these agents. There has also been a need to provide agents which enable cytotoxic substances to be used in increased doses in order to increase their effectiveness.

It was therefore an object of the present invention to provide an agent for the selective chemoprotection of human cells, in particular human bone marrow progenitor cells and to provide stem cells that make it possible to protect healthy cells from the damaging side effects of cytotoxic agents.

This object is achieved by the use of L-cysteine or / and derivatives thereof for the production of a pharmaceutical composition for the selective protection of non-malignant cells during a therapy for inducing cell death of malignant cells.

It was surprising that the use according to the invention of L-cysteine or / and derivatives thereof reduces the negative side effects of agents for inducing cell death in malignant cells, but has no inhibitory effect on their effect in inducing cell death in malignant cells. In particular when carrying out ex vivo treatments, the use of L-cysteine and / or derivatives thereof according to the invention shows a strong reduction in the damage to non-malignant cells caused by chemotherapeutic agents. The method according to the invention not only has the great advantage that the cytotoxic damage to non-malignant cells is reduced, at the same time there can be a synergistic effect with the agent used to induce cell death in malignant cells. The agent used according to the invention preferably not only protects the non-malignant cells, but can even improve their proliferation. This can increase the number of non-malignant cells after the malignant cells have been killed.

Particularly in the case of cell types that are only present in a small proportion in a tissue, such as, for example, bone marrow precursor / stem cells, the number of non-malignant cells that can be re-implanted in a patient can be significantly increased in an ex vivo treatment. Reimplanting a larger amount of healthy cells increases the likelihood of a successful course of treatment.

In a preferred embodiment, the therapy for inducing cell death of malignant cells comprises chemotherapy. However, there are also other methods known in the field for inducing cell death of malignant cells, such as intermittent chemotherapy, shock therapy, low-dose long-term therapy, hypertherapy, radiation therapy or nuclear medicine methods. The therapy particularly preferably comprises the administration of a cytostatic from the group of the alkylating agents, such as, for example, cyclophosphamide, trofosfamide, ifosfamide, chlorambucil, melphalan, carmustine, lomustine, semustine, busulfan, cisplatin, in particular the DNA crosslinker. Most preferably, cis-platinum or / and carbo-platinum are used as cytostatic agents. The pharmaceutical composition used according to the invention preferably contains L-cysteine as the active ingredient. Furthermore, the pharmaceutical composition can contain physiologically active derivatives of L-cysteine, to which N-modified derivatives such as N-acyl compounds such as N-acetyl-L-cysteine, carboxyl-modified derivatives such as esters or amides or S- modified derivatives, such as S-alkylated compounds. The SH group is preferably freely available. Derivatives of L-cysteine also include precursor substances which are reacted in the organism or in the extracorporeal liquid containing cells to be treated and which form or release L-cysteine and / or a physiologically active derivative thereof.

L-cysteine is reabsorbed in the kidney and is available to the body again. Active substances are therefore preferred which behave similarly to L-cysteine with regard to their pharmacokinetic properties. It is therefore advantageous with corporeal treatment that the dose of the substances used according to the invention can be kept low due to this "natural recycling process". This is one way of reducing costs.

For the corporeal treatment of an organism, the pharmaceutical composition can be brought into various galenical forms, such as tablets, coated tablets, solutions for injection or infusions. The administration is systemic or local depending on the requirements. The dose of the compound used according to the invention is adjusted so that the chemotherapeutic agent has an optimal effect with the best possible chemoprotective protection by the agent according to the invention.

The dosage of the pharmaceutical composition is preferably from 0.1 to 1000 mg L-cysteine or a physiologically active derivative thereof per kg body weight, particularly preferably from 1 to 400 mg / kg body weight.

The dosage of the cytostatic is adjusted in each case and generally depends on the cytostatic used, the area of application and the form of therapy used. The use of L-cysteine or a physiologically active derivative thereof can reduce or largely avoid the side effects which occur at a customary dosage level of the cytostatic. Surprisingly, it is also possible to increase the dose of the cytostatic and thus its effectiveness without having to accept an increase in side effects at the same time.

With various cytostatics, their effectiveness decreases with prolonged use. To counteract a reduction in effectiveness, the dose used must therefore be increased as treatment progresses. This increase in dose is countered by the side effects mentioned. The dose used can therefore only be increased up to a certain maximum limit. The use of the agent according to the invention can surprisingly increase the dose of the cytostatic. As a result, the malignant cells can be eliminated more effectively and the time-related loss of activity of the cytostatic agent can be counteracted.

In a preferred embodiment, the therapy comprises extracorporeal treatment of cells. The cystostat used is used in suitable doses to achieve optimal induction of cell death in malignant cells. Cis-platinum or / and carbo-platinum is preferably used in a concentration of 0.3 to 30 μg / ml in a suspension containing cells to be treated.

The active ingredient used according to the invention is used in a concentration of 0.01 mg / ml to 4 mg / ml in a suspension containing the cells to be treated. The active ingredient used is preferably L-cysteine in a concentration of 0.01 mg / ml to 3 mg / ml, particularly preferably 0.1 mg / ml to 1.5 mg / ml and most preferably 0.2 mg / ml to 1 mg / ml used. The N-acetyl-L-cysteine used as active ingredient is preferably in a concentration of 0.1 mg / ml to 4 mg / ml, particularly preferably 0.5 mg / ml to 2 mg / ml, most preferably 0.9 mg / ml to 1.5 mg / ml used.

The active ingredient can be added before, during or / and after the addition of the cell death inducing agent. The extracorporeal treatment of the cells can be carried out at any suitable temperature when the selective-protective pharmaceutical composition is used according to the invention, preferably the treatment is carried out at a temperature of from 20 ° C. to 42 ° C., preferably at 37 ° C.

According to the invention, the active ingredient can be used in therapy methods for inducing the cell death of malignant cells of any known cell type. The cells to be treated are preferably selected from peripheral blood lymphocytes, bone marrow cells, Subpopulations thereof and / or hematopoietic stem cells, preferably from bone marrow. The cells to be treated preferably originate from a human patient with a malignant disease. After an extracorporeal treatment, the cells can be re-implanted in the patient.

In a preferred embodiment, the use of the active ingredient according to the invention is carried out in a treatment under sterile conditions in plastic vessels, in particular in one or more flexible plastic vessels such as blood bags. The plastic containers are preferably closed and have at least one opening e.g. an extension for the connecting hoses. Via these connecting hoses, which preferably consist of weldable plastic material, the connection and disconnection of bags is possible with a suitable device in such a way that a closed system is always maintained. This means that the handling of biological substances such as cells, viruses or vectors is possible with a significantly reduced risk of contamination and the use of sterile workbenches can even be dispensed with.

This makes it possible for blood to be taken from a patient via hose connections, for example under sterile conditions, and for all extracorporeal treatment steps for the cells to take place in this system. These treatment steps include incubating, cultivating, introducing and removing liquids or cells from the plastic container, as well as genetic engineering process steps. If necessary, the plastic container can be coupled to a further plastic container containing solutions, cells, vectors, etc., and the two volumes can thus be combined. Reactions such as transduction can then take place in this combined volume. After completion of the extracorporeal treatment, the cells can be concentrated by centrifugation and transferred to a solution suitable for reimplantation, which can be buffered if necessary. The thus obtained cell suspension can turn ^'are supplied to the patient via hose connections sterile. With regard to the plastic vessels and their use, reference is expressly made to WO 98/07829.

In addition, the extracorporeal treatment can also include gene therapy process steps. These include, for example, transduction of the cells, for example using retroviral vectors, stimulation of the cells, cultivation of the cells and / or extraction of cell subpopulations. In a further embodiment, the invention relates to a method for the selective protection of non-malignant cells in a therapy for inducing cell death of malignant cells in a living being, which is characterized in that the living being is given a pharmaceutical composition comprising L-cysteine and / or derivatives thereof in a effective amounts for the selective protection of non-malignant cells.

The following examples, publications and the illustrations further explain the invention, the scope of which results from the patent claims. The described methods are to be understood as examples which describe the subject matter of the invention even after modifications.

The illustrations show the following:

Fig. 1 Anti-proliferation and chemoprotective effects of the compounds

Glutathione (GSH), L-cysteine (L-CYS) and N-acetyl-L-cysteine (NAC) on PBL (peripheral blood lymphocytes) after activation with PHA (phythaemagglutinin) and 72 h incubation. CDDP concentration: 3μg / ml. The y-axis zero value represents the CDDP control. The values for the relative proliferation are calculated from the quotients of proliferating cells to non-proliferating cells (BrdU / Hoechst cell cycle analysis).

Fig. 2 De novo effect or chemoprotective effect of the compounds GSH,

NAC and L-CYS on CD34 ⁺ bone marrow cells (KM) cells sorted according to CD38 ⁺ and CD38 ^" . Initial cell number 100. The compounds are added in a concentration of 0.5 mg / ml and 1 mg / ml, respectively. CDDP concentration: 3 μg / ml.

Fig. 3 Protective effect of the compounds GSH, NAC and L-CYS (1 mg / ml) after delayed addition to CDDP (3 μg / ml) treated KM cells. The y-axis zero value represents the CDDP control. The CD34 ⁺ KM cells were counted after 14 days of culture using digital image analysis and trypan staining. Starting cell count: 100 cells / well. Examples

To demonstrate the protective effect of L-CYS and NAC, their chemoprotective and proliferation-inhibiting or proliferation-increasing effects on peripheral blood lymphocytes (PBL) and on bone marrow cells (CD34 ⁺ ) and on subpopulations thereof (CD34 ⁺ CD38 ⁺ and CD34 ⁺ CD38 ^" ) are examined GSH is used as a reference substance.

At the time of toxification with 3 μl / ml CDDP, the test substances GSH, L-CYS or NAC in the concentrations 0.1, 0.5 and 1.0 mg / ml are added to the cell lines and incubated for 24 h. The Brdü / Hoechst cell cycle analysis used for the proliferation analysis yielded the results shown in the following examples.

1. Material and methods

1.1 cells

1.1.1 Human peripheral blood lymphocytes

Human PBL's are obtained from fresh, heparinized whole blood from healthy, adult donors using standard Ficoll isolation. The pellet obtained is in 1 ml of RPMI 1640 culture medium; 15% FCS; 1% autologous donation plasma; 2 raM glutamine; non-essential amino acids (each 0.1 mM); 1 mM sodium pyruvate; 2-10 ^"5 alpha-thioglycerol; activation with 5 μg / ml PHA for Brdü / Hoechst cell cycle analysis supplemented with 8-10 ^{" 5} M deoxycytidine and 8-10 ^"5 M Brdü resuspended and the cell number determined by means of Tyφan blue vital staining.

1.1.2 Human bone marrow cells

Human bone marrow from sternal punctates from donors between the ages of 20 and 60 serves as the starting material. After working up, Ficoll isolation and resuspending, the bone marrow is placed in freezing medium Isove's DMEM; 10% FCS, 10% DMSO; 2 mM glutamine; 100 IU / ml penicillin; 1 mg / ml streptomycin stored at -196 ° C in liquid nitrogen. Cultivation of bone marrow cells

After thawing, the CD34 + bone marrow populations are presented with a cell count of 50 or 100 cells in 96-well round-bottom plates with 100 μl of Iscove's culture medium (Iscove's DMEM; 20% human AB plasma or 20% FCS; 2 mM glutamine; 100 IU / ml penicillin; 1 mg / ml streptomycin) sorted using a cell sorter. The medium of the KM cells is supplemented with a cytokine cocktail (IL3 250 U / ml; IL6 1000 U / ml; EPO 5 U / ml; GM-CSF 100 U / ml; SCF 10 U / ml). After 7 days of incubation at 37 ° C. and 7% CO ₂ , the cells are fed with 50 μl of Iscove's culture medium, supplemented with the cytokine cocktail.

1.2 reporter molecules

The DNA-specific fluorochromes and the fluorochromes of Table 1 coupled to monoclonal antibodies (MAK) are used.

Table 1

MAK (anti-human mouse antibody) conjugated to fluorochrome and its main cell specificity

Table 2

Devices and filter combination for the flow cytometric methods used

Results analysis software: Lysis2 (Becton Dickinson) MPLUS (Phoenix Flox Systems) Excel 5.0 (Microsoft)

1.3 Verification procedure

1.3.1 BrdU / Hoechst cell cycle analysis

Differential cell cycle analyzes of up to three consecutive cell cycles are determined with the help of the BrdU / Hoechst technique. The analysis is carried out according to the manufacturer's instructions and as in Kubbies, M. (1992), High-resolution cell cycle analysis: the flow cytometric bromodeoxyuridine-hoechst quenching technique, in: A. Radbruch (ed.): Flow cytometry and cell sorting, Springer Verlag, Berlin, pp. 75-85.

1.3.2 Proliferation studies of human peripheral blood lymphocytes

Peripheral blood lymphocytes (PBL) are proliferatively in a resting phase (Go phase). The lymphocytes are stimulated with PHA (phytohemagglutinin) and begin to proliferate asynchronously after about 30-40 h in the S phase of the first cell cycle. The lymphocytes are with a cell density of 3-10 ⁵ cells per ml BrdU medium RPMI 1640, 15% FKS, 1% autologous donation plasma (whole blood centrifuged for 10 min at 400xg), 2-10 ^"5 M alpha-thioglyerol, 8- 10 ^"5 M BrdU, 8-10 ^{" 5} M deoxycytidine are sown in 24-well plates with 2 ml medium per well. The alpha-thioglycerol added to the medium serves to improve the proliferation of PBL (Kubbies, M. et al., Lymphokine Res. , 9 (1990), Improvement of human lymphocyte proliferation and alteration of IL-2 secretion kinetics by alpha-thioglycerol, 95-101), deoxycytidine prevents possible nucleotide metabolism disorders which can be caused by BrdU.

After adding the chemotherapeutic agent and the substance according to the invention or the comparative substance GSH, the cells are stimulated with 5 μg / ml PHA. In the time delay tests, the test substances are added at a later time.

The cells treated in this way are incubated for 72 h, then centrifuged at 250 × g, 10 min and then stained.

1.3.3 Hoechst / PI (propidium iodide) staining

The samples frozen at -20 ° C are thawed and centrifuged at 250xg for 10 min. The pellet is resuspended in 10 ul RNAse A 1000 U / ml. This degrades the RNA, which would otherwise be stained by PI. The resuspended cells are now in 1 ml DNA staining buffer 100 mM Tris 7.4, 154 mM NaCl, 1 mM CaCl ₂ , 0.5 mM MgCl ₂ , 0.2% BSA, 0.1% NP40 with 1 μg / ml HO33258 and incubated for 15 min at 4 ° C in the dark (approx. 0.5 - 1.0 - 10 ⁶ cells / ml). Subsequently 2 μg / ml PI are added and incubated again at 4 ° C. for 15 min. The samples colored in this way can be measured within 8 hours when stored in the dark at 4 ° C.

1.3.4 Evaluation

The evaluation is carried out with the help of the program MPLUS, as in Kubbies, M. (1992), High-resolution cell cycle analysis: the flow cytometric bromodeoxyuridine-hoechst quenching technique, in: A. Radbruch (ed.): Flow cytometry and cell sorting , Springer Verlag, Berlin, pp. 75-85.

The degree of inhibition of proliferation is determined from the quotient of the number of proliferating cells by the number of non-proliferating cells (see formulas). Of each The result is that the value of the cis-platinum control is subtracted so that it represents the y-axis zero value. The results are standardized with the normalization factor (for PBL in the experiment "CDDP (cis-platinum) protection" = 5.5, in the experiment "time delay" = 1.35) so that the control receives the relative proliferation value 10 (> 10 = Stimulation; <10 = inhibition). The size of the nomination factor allows a statement about the prohferative state of the cells. The smaller the value, the more cells are in a prohferative stage after the end of the culture period. For the PBL, the value indicates the donor-dependent stimulability of the cells by PHA. The smaller the value, the higher the number of cells that can be stimulated by PHA.

Formulas:

Synchronous cell populations Asynchronous cell populations

RP: relative proliferation; CP: value determined for the cis-platinum control; % Rx: percent of cells in this region, all regions together => 100%. NF: normalization factor.

1.3.5 Staining of the bone marrow

After thawing and overnight culture in 24-well plates, the bone marrow cells are pooled and the cell number of the mononuclear cells is determined by means of trypan blue vital staining. To determine the autofluorescence, 1-5-10 ⁵ cells are removed and the remaining cells are stained. The cells are centrifuged at 250xg for 10 min. The supernatant is then discarded and the cells in 100 μl sterile-filtered staining medium (washing medium with antibody, Iscove's DMEM; 10% FCS; 2 mM glutamine; 100 IU / ml penicillin; 1 mg / ml streptomycin) per 10 ⁶ cells and resuspended and 20 min without direct light irradiation with the fluorescence-labeled MAK (antibodies: per 10 cells, 100 ul supplemented with 20 ul <CD34> -FITC, 20 ul <CD38> -PE and 5 ul <HLA-DR> -TC) are incubated at room temperature. The mixture is then centrifuged at 250xg for 10 min and washed the pellet with washing medium. The cells are taken up in a density of approx. 10 cells / ml in washing medium and sorted.

1.3.6 Sorting of the bone marrow cells

The cells are sorted using a scattered light and antibody gate. The first gate in the scattered light image is set so that only living cells are sorted. With a logical AND link, the gates are set in the second step in the 2-dimensional fluorescence image of the FITC and PE fluorescences so that the <CD34 ⁺ > KM cells can be sorted into <CD38 ⁺ > or <CD38 populations. In a third picture, the <CD34 ⁺ > cells are analyzed for their <HLA-DR> properties, but not sorted according to this.

1.3.7 Digital image analysis

The KM cells cultured in the 96-well plates with a round bottom are counted with an inverted microscope and the Quantimet 570 image analysis system (Cambridge Instruments, Leica) IM (Zeiss).

2 results

2.1. Chemoprotection of human peripheral blood lymphocytes

Human PBL is used as a model for normal, diploid, hematopoietic cells. As such, GSH, NAC and L-CYS have no proliferation-inhibiting effects at the doses of 0.1 mg / ml, 0 ₃ 5 mg / ml and 1 mg / ml used. The simultaneous use of CDDP as a cytostatic and 0.1 mg / ml GSH, L-CYS or NAC only leads to slight protection with GSH with a relative proliferation of approx. 25% of the control value. At a dose of 0.5 mg / ml and 1 mg / ml, L-Cys and NAC show significantly better CDDP protection than the comparison substance GSH. The protective effect of L-Cys at 0.5 mg / ml is significantly higher than that of GSH.

At a concentration of 1 mg / ml, the values of the relative proliferation are between 55% (NAC) and 82% (L-CYS) of the control values (see Fig. 1 as an example). 2.2. Chemoprotection of human CD 34 ⁺ bone marrow drug sites

In order to investigate the chemoprotective protection of the substances used according to the invention on the heterogeneous population of the CD34 ⁺ -KM progenitor cells, these were sorted into two phenotypic populations namely CD38 ⁺ and CD38 ^" -KM cells. The CD34 ⁺ / CD38 ^" cell population represents hardly differentiated, very early KM progenitor cells / stem cells. The CD34 ⁺ / CD38 ⁺ cell population mainly contains CFU cells (colony forming units) and among other things already myeloblasts, erythroblasts and lymphoblasts (Terstappen, WMM, et al ., Blood 77 (1991) 1218-1227).

In order to be able to carry out a further analytical differentiation of the sorted cells, the presence of the activation-correlated histocompatibility-antigen HLA-DR is additionally determined by flow cytometry. The proportion of CD34 ⁺ cells in the total of mononuclear cells averaged 1.2% (± 0.3, n = 6). The proportion of the CD38 ⁺ , HLA-DR ⁺ cells was on average 73% (± 7.0), the CD38 ⁺ , HLA-DR ^" cells 4% (± 1.1), the CD38 ^" , HLA-DR ⁺ cells 20% (± 5.5) and the CD38 ^" , HLA-DR ^" cells 3% (± 0.7).

A cell count of 50 or 100 KM cells is sorted into 96-well round-bottom plates and then L-CYS, NAC or the comparative compound GSH and CDDP are simultaneously added to the culture medium. With a cultivation period of 14-16 days, the cells are counted with the help of digital image analysis and additionally with trypan blue vital staining and counting chamber.

Fig. 2 shows the results of cell proliferation and chemoprotection of cells from a representative KM donor. A slight variation in proliferation can generally be seen in different donors.

A comparison of the cell counts on day 15 after the start of the treatment most clearly illustrates the different effectiveness of the test substances.

KM CD34 ⁺ CD38 ⁺ cells:

The comparison substance GSH cannot protect the KM cells from the cytotoxic effects of CDDP neither at a concentration of 0.5 mg / ml nor at 1 mg / ml. Even the gift of GSH alone without CDDP leads to a reduction in the number of cells to approximately half (0.5 mg / ml GSH) or a cell number that almost corresponds to the negative control (CDDP).

L-CYS, on the other hand, has a strong protective effect at a concentration of 1 mg / ml. The cell number with simultaneous administration of CDDP and L-CYS (1 mg / ml) is approx. 70% of the control value. L-Cys alone does not lead to a significant reduction in the number of cells compared to the control value. In contrast, NAC had no protective effect.

KM CD34 ⁺ CD38 cells:

In this case, the comparative substance GSH shows little protective effect against CDDP. When CDDP and GSH are administered simultaneously, only approx. 15% (1 mg / ml GSH) or approx. 25% (0.5 mg / ml GSH) of the cell number are available compared to the control. The administration of GSH alone without CDDP leads to a reduction in the number of cells to approximately half (0.5 mg / ml GSH) or a number of cells that almost corresponds to the negative control (CDDP).

In contrast, with a dose of 0.5 mg / ml NAC, 1 mg / ml NAC or 1 mg / ml L-CYS the number of cells is 50% higher, the same number of cells or an increase of approx. ) compared to the control. Both NAC and L-CYS show a strong protective effect against CDDP when administered simultaneously with CDDP in all concentrations used. With the different concentrations of the active substances used, approximately 70 to 55% of the cells are protected from the cytotoxic effect of the CDDP.

2.3. Chemoprotection with delayed addition of the protective substances

With the time-delayed addition of the substances L-CYS and NAC used according to the invention to cells treated with CDDP, it could be shown that the chemoprotective effect is not based on a direct reaction with CDDP in the culture medium (see FIG. 3).

In the case of the bone marrow cells tested, a strong growth improvement by L-CYS can already be observed during the substance controls, while GSH and NAC have an inhibitory effect on the CD34 ⁺ KM progenitor cells. With the addition of GSH and NAC, only a slight protection of the cells can be seen at the 0 h value and at 14 h no significant protection can be seen in comparison to the CDDP control. L-CYS, on the other hand, shows a good protective effect at 0 h, 1 h and 4 h (about 60% of the cell number of the control when added) after 4 h). Even with L-CYS administration after 14 h, approx. 25% of the cells can still be protected against the cytotoxic effects of CDDP.

Table 3 Summary of the chemoprotective effects of the test substances

* not an object of the invention ++ good to complete protection + light protection - no protection

The results of the tests are summarized again in Table 3. It can be seen from this that the substances NAC and L-CYS used according to the invention show both proliferation-increasing and chemoprotective properties in the treatment of KM cells and peripheral blood lymphocytes (PBL).

There is also a superiority in the proliferation-increasing and chemoprotective effect of the substances NAC and L-CYS used according to the invention compared to the substance GSH known from the prior art.

Reference list

Civin, C. L, Csore, S.D., J. Hematotherapy 2 (1993) 137-144

Kubbies, M. (1992), High-resolution cell cycle analysis: the flow cytometric bromodeoxyuridine-hoechst quenching technique, in: A. Radbruch (ed.): Flow cytometry and cell sorting, Springer Verlag, Berlin, pp. 75-85

Kubbies, M. et al., Lymphokine Res., 9 (1990)

Kubbies, M., et al., Br. J. Cancer 64 (1991) 847-849

Terstappen, W.M.M., et al., Blood 77 (1991) 1218-1227

WO 98/07829

Claims

claims

1. Use of L-cysteine or / and derivatives thereof for the production of a pharmaceutical composition for the selective protection of non-malignant cells during a therapy for inducing cell death of malignant cells.

2. Use according to claim 1, characterized in that the therapy comprises chemotherapy.

3. Use according to one of claims 1 or 2, characterized in that the therapy comprises the administration of a cytostatic from the group of alkylating agents, in particular the DNA crosslinker.

4. Use according to claim 3, characterized in that cis-platinum or / and carbo-platinum is used as the cytostatic.

5. Use according to one of claims 1 to 4, characterized in that the pharmaceutical composition contains L-cysteine and / or N-acetyl-L-cysteine as an active ingredient.

6. Use according to one of claims 1 to 5, characterized in that the therapy comprises an extrakoφoreal treatment of cells.

7. Use according to claim 6, characterized in that cis-platinum or / and carbo-platinum is used in a concentration of 0.3 to 30 μg / ml in a suspension containing the cells to be treated.

8. The method according to claim 6 or 7, characterized in that the active ingredient is used in a concentration of 0.01 mg / ml to 4 mg / ml in a suspension containing the cells to be treated.

9. Use according to claim 8, characterized in that the active substance L-cysteine in a concentration of 0.01 mg / ml to 3 mg / ml, preferably from 0.1 mg / ml to 1.5 mg / ml, particularly preferred 0.2 mg / ml to 1 mg / ml is used.

10. Use according to claim 8, characterized in that the active ingredient is N-acetyl-L-cysteine in a concentration of 0.1 mg / ml to 4 mg / ml, preferably 0.5 mg / ml to 2 mg / ml most preferably from 0.9 mg / ml to 1.5 mg / ml is used.

11. Use according to one of claims 6 to 10, characterized in that the treatment at a temperature of 20 ° C to 42 ° C, preferably at 37 ° C.

12. Use according to one of the preceding claims, characterized in that the cells to be treated are selected from peripheral blood lymphocytes, bone marrow cells, subpopulations thereof or / and hematopoietic stem cells, preferably from bone marrow.

13. Use according to one of claims 6 to 12, characterized in that the cells to be treated come from a human patient with a malignant disease.

14. Use according to claim 13, characterized in that the cells are reimplanted to the patient after the treatment.

15. Use according to one of claims 6 to 14, characterized in that the treatment is carried out under sterile conditions in one or more flexible plastic containers.

16. Use according to one of claims 6 to 15, characterized in that the extracoφoreal treatment further comprises gene therapy process steps. .

17. Use according to claim 16, characterized in that the gene therapy process steps comprise transduction of the cells.

18. Use according to claim 17, characterized in that the cells are transduced with viral vectors.

19. The method according to any one of claims 16 to 18, characterized in that the gene therapy process steps include stimulation and / or cultivation of the cells.

20. Extrakoφoreales method for the selective protection of non-malignant cells during therapy for induction of cell death of malignant cells, characterized in that L-cysteine or / and a derivative thereof is added before, during and / or after the therapy of the cells.

21. A method for the selective protection of non-malignant cells during a therapy for inducing the cell death of malignant cells in a living being, characterized in that the living being is given a pharmaceutical composition comprising L-cysteine or / and derivatives thereof in an amount effective for the selective protection of non-malignant cells administered.